The Small Ubiquitin-Related Modifier (SUMO) pathway modulates protein activity through dynamic and reversible conjugation of SUMO to substrates. Among the targets of SUMO post-translational modification are a number of transcriptional regulators and proteins that play vital roles in maintaining genome integrity both at the level of chromosome packaging, cohesion, and segregation as well as at the level of DNA synthesis, repair, and homologous recombination. Although SUMO conjugation is known to modulate protein interactions, the functional consequences of SUMO modification are only beginning to be understood. In contrast to previous beliefs, recent studies have suggested that SUMO conjugation can result in protein ubiquitylation and subsequent proteasomal degradation. A newly identified class of ubiquitin ligases referred to as SUMO-targeted ubiquitin ligases (STUbLs) putatively mediate the ubiquitylation of SUMO conjugates. These proteins physically interact with SUMO and mediate the ubiquitylation of proteins containing tandem SUMO-like domains (SLDs), and deletion of the heterodimeric STUbL in S. cerevisiae results in an accumulation of high molecular weight SUMO conjugates. The latter two observations suggest that SUMO chains, the physiological function of which is presently unknown, may act as the signal for protein degradation. Critically, however, STUbL mediated ubiquitylation of a physiologically relevant SUMO conjugated substrate has never been demonstrated. The long-term objective of the research described in this proposal is to elucidate the mechanism by which SUMO conjugation regulates protein stability via the ubiquitin proteasome pathway. Specifically, studies will seek to clarify the role of STUbLs in targeting SUMO conjugates for ubiquitylation. STUbLs from three organisms, S. pombe, S. cerevisiae, and human, will be utilized in biochemical and crystallographic studies designed to characterize the interaction of STUbLs with SUMO conjugated or tandem SLD containing proteins in an effort to define the mechanism of STUbL activity. The specific aims of this research are to: 1) Characterize the structural and biochemical basis for STUbL mediated ubiquitylation of proteins containing tandem SLDs and 2) Reconstitute and characterize the structural and biochemical basis of STUbL mediated ubiquitylation of a SUMO conjugated substrate. PUBLIC HEALTH RELEVANCE The importance of this research to health is suggested by the requirement of a properly functioning SUMO pathway for cell viability, the functional diversity of SUMO substrates, and the involvement of SUMO in all stages of development of multi-cellular organisms. An understanding of how SUMO regulates the activities, interactions, and stabilities of cellular proteins is vital for understanding cell function in health and disease.